Developing an effective vaccine against HIV/AIDS remains an important global health target to inhibit and reverse the high level of HIV-1 transmission currently ongoing around the world. To date, there have been four large HIV-1 vaccine trials, of these only RV144 exhibited a limited, but significant protection (31.2%) from HIV- 1 acquisition. The assessment of the results from this study has revealed a number of key correlates of protection, including antibodies that target a conformational epitope in the V1V2 region of Env. It is clear that new vaccination methodologies are needed to improve upon the findings in the RV144 trial by generating high titers of V1-V2 and functional antibodies, creating long lived B cell memory, and driving antibody maturation toward broadly neutralizing activity. The oral mucosal tissues provide a unique environment in which to vaccinate, owing to its accessible lymphoid organs, high density of immune cells and increased potential for generating a mucosal response that is able to prevent HIV-1 acquisition at a mucosal surface. We propose a vaccination strategy to deliver recombinant HIV-1 trimeric Envelope (Env) protein vaccines directly to the oral mucosa (buccal mucosa of the cheek) by intra-epithelial (IEp) vaccination. Vaccines will be tested with two adjuvant formulations designed to stimulate strong innate induction through TLR4 and TLR7/8 pathways. The goal of this proposal is to optimize oral mucosal vaccination of the HIV-1 Env protein in order to enhance Env- specific antibody responses, and to characterize the effect of oral vaccination with Env by conducting a thorough multi-parametric assessment of vaccine-elicited immunity. The first Specific Aim will assess the innate immune response to the vaccine, with a focus on the influence of the vaccine at the buccal mucosa where the vaccine is administered by IEp injection. Innate immune changes are interesting for their potential to reveal a biomolecular signature that we believe will correspond to superior HIV-1 ENV antibody responses. The second and third Specific Aims will evaluate whether supplying Env to the oral mucosa drives superior antibody responses at mucosal sites and systemically. An assessment of the neutralization and ADCVI activity will provide a functional assessment that can be correlated with the findings from studies of the innate immune response. Aims 2 and 3 will also involve a mechanistic assessment of the anti-Env antibody response through an assessment of both CD4 T Follicular Helper cell responses and antibody maturation in these macaques. In addition to the optimization of oral mucosal IEp vaccination and the exhaustive multi-parametric analyses, the combined expertise of the Sodora and Sather laboratories is one of the major strengths of this application. The experiments outlined in this proposal utilize an oral mucosal IEp delivery of next-generation HIV-1 Env antigens, as well as an assessment of both innate and adaptive immune responses. This comprehensive approach results in a high degree of confidence that undertaking these studies will make a significant contribution to HIV-1 vaccine development and advance efforts toward reversing the ongoing HIV-1 pandemic.